Pollen production in flowering plants is a highly regulated developmental process which occurs within the diploid sporophytic tissue of the anther. The normal development of the male gametophyte is dependent upon the tapetum which lines the locular space of the anther. The tapetum is thought to provide the developing microspore/pollen with nutrients and other necessary products such as enzymes and structural components (Pacini et al., Plant Syst. Evol., 149:155-185, 1985). The importance of the role of the tapetum is illustrated by the fact that male sterility can result from tapetal malfunction (Kaul, Male Sterility in Higher Plants, 1988; Koltunow et al., Plant Cell, 2:1202-1224, 1990; Mariani et al., Nature, 347:737-741, 1990).
In Brassica, the secretory tapetum is composed of cells which are metabolically very active until approximately microspore mitosis at which time they degenerate (Grant et al., Can. J. Bot., 64:779-786, 1986; Murgia et al., Sex Plant Reprod., 4:28-35, 1991; Polowick and Sawhney, Sex Plant Reprod., 3:263-276, 1990). Their cellular contents such as lipids are then released into the anther locule where they are thought to contribute to the formation of the external pollen coat (Evans et al., Planta, 186:343-354, 1992; Heslop-Harrison, New Phytol, 67:779-786, 1968). In the Brassica napus cv. Westar used in the present invention, the tapetal lipids accumulate in small lipid bodies of less than 1 .mu.m in diameter which are either free or in membrane-bound clusters (Polowick and Sawhney, Sex Plant Reprod., 3:263-276, 1990). Membrane-bound oil bodies of similar size have been well characterized in seed storage tissues where they were found to be associated with specialized proteins called oleosins (Huang, Ann. Rev. Plant Physiol. Plant Mol. Biol., 43:177-200, 1992; Murphy, Prog. Lipid Res., 32:247-280, 1993). Oleosins are believed to help stabilize the small oil bodies during dehydration and possibly provide binding sites for lipase activity upon germination. Recently, a novel class of oleosins was shown to be associated with the lipid bodes of B. pollen grains where they are believed to serve a similar function (Roberts et al., Plant J., 3:629-636, 1993). Putative oleosins have also been reported in the anthers of Arabidopsis thaliana (De Oliveira et al., Plant J., 3:495-507, 1993).
Two highly homologous Brassica napus flower cDNA clones, Sta 41-2 and Sta 41-9, were isolated and characterized (Robert et al, Plant J., 1994, 6:927-933). These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41-2 and Sta 41-9 proteins possessed characteristics similar to oleosins such as a polar N-terminal domain, a large relatively conserved hydrophobic domain and a long C-terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41-2 and Sta 41-9 proteins have a basic pI, lack of a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.
Tapetal-specific cDNAs have been isolated from Anthirrhinum majus (Nacken et al., Mol. Gen. Genet. 229:129-136, 1991; Nacken et al., FEBS Lett. 280:155-158, 1991), Brassica napus (Scott et al., Plant Mol. Biol. 17:195-207, 1991; Shen and Hsu, Mol. Gen. Genet. 234:379-389, 1992; Hird et al., Plant J. 4:1023-1033, 1993; Robert et al., Plant J. 6:927-933, 1994), Sinapis alba (Staiger and Apel, Plant J. 4:697-703, 1993), maize (Wright et al., Plant J. 3:41-49, 1993), tobacco (Koltunowetal., Plant Cell 2:1201-1224, 1990), tomato (Smith et al., Mol. Gen. Genet. 222:9-16, 1990) and rice (Tsuchiya et al., Plant Mol. Biol. 26:1737-1746, 1994). The identity and/or function of the products encoded by most of these tapetum-specific transcripts are unknown.
Only three tapetum-specific genes and their regulatory sequences have been studied in detail: TA29 from tobacco (Koltunow et al., Plant Cell, 2:1201-1204, 1990; Mariani et al., Nature, 347:737-741, 1990), tapl from Antirrhinum majus (Spena et al., Theor, Appl. Genet. 84:520-527, 1992; Nacken et al., Mol. Gen. Genet. 229: 129-136, 1991) and A9 from Arabidopsis thaliana (Paul et al., Plant Mol. Biol. 19:611-622, 1992). However, the cis elements required for tapetal gene expression are still not known. The temporal expression pattern of the Sta 41 promoter was somewhat different from the above mentioned tapetalspecific promoters, which were all expressed during the early stages of pollen development from meiosis to late microspore interphase or early mitosis, corresponding well with the timing of tapetum cell differentiation and degeneration. The temporal expression pattern of the Sta 41 promoter was evident from the uninucleate microspore stage to the early trinucleate stage in pollen development, by which time the tapetum had exhibited considerable signs of degeneration (Grant et al., Can. J. Bot., 64:779-786, 1986; Murgia et al., Sex Plant Reprod., 4:28-35, 1991; Polowick and Sawhney, Sex Plant Reprod., 3:263-276, 1990).